The present invention relates to a rotary thermocycling apparatus, and to methods of using rotary thermocycling apparatus, especially for use in biochemical reactions and in particular for use in polymerase chain reactions (PCR). In embodiments, the present invention is directed to rotary thermocycling processes in which samples are placed on filters, or other media, and heated on plates in a sequence at predetermined temperatures, preferably including at least one step in which the sample is sprayed e.g. with liquid reagent(s), during each cycle.
The present invention w ill be particularly described herein with respect to biochemical reactions.
It is frequently necessary or desirable to be able to quantify and/or detect the presence of certain nucleic acid molecules or microorganisms in samples of air, soils, water, food, body fluids and other materials. This may be necessary in relation to an immediate or health situation, or in testing to determine safety for use by humans or animals. Thus, in many instances, it is important to be Able to accurately and quickly confirm the presence and quantity of, or absence of, particular microorganisms in samples, and to do so in an automated, reliable and reproducible manner.
Traditional quantitative estimates of microorganisms in medical, food, environmental and other samples were based on colony counts after suitable culturing in diluted samples on nutrient agar plates. However, more accurate and rapid detection of microorganisms in various types of test samples has become possible.
Genetic information in al living organisms is carried largely in nucleic acids, either double-stranded deoxyribonucleic acids (DNA) or ribonucleic acid (RNA), and detection and discrimination on the basis of specific nucleic acid sequences has permitted the detection of the presence or absence of a particular organism within a test sample. The development of the polymerase chain reaction (PCR) process for amplifying one or more targeted nucleic acid sequences within a sample has greatly facilitated processes for detecting and discriminating specific nucleic acid sequences, and hence specific organisms.
PCR methods of detection require multiple or cyclic chemical reactions to produce a desired product, under carefully controlled temperature conditions to ensure accuracy and reproducibility, in order to produce sufficient material to enable detection of a microorganism in the sample, or indicate absence of the microorganism. Apparatus and methods have been developed which permit the accurate control of the temperature of reaction vessels in which such PCR amplification reactions may be performed. For example, there are a number of thermocyclers used for DNA amplification and sequencing in which one or more temperature controlled elements or xe2x80x9cblocksxe2x80x9d hold samples containing the reaction mixture, and the temperature of the block is varied over time. In other systems, a robotic arm is used to move mixtures from one block to another. These systems include features which allow the user to program temperatures or temperature profiles of the block over selected periods of time so that various processes e.g. DNA denaturing, annealing and extension, can be efficiently accomplished.
Polymerase chain reaction (PCR) is a technique involving multiple cycles that results in geometric amplification of certain polynucleotide sequences each time a cycle is completed. The technique is now well known. One example of PCR involves Denaturing a double-stranded polynucleotide, followed by annealing at least a pair of primer oligonucleotides to the resultant single-stranded polynucleotides. After the annealing step, an enzyme with polymerase activity catalyzes synthesis of a new polynucleotide strand that incorporates the primer oligonucleotide and uses the original denatured polynucleotide as a synthesis template to produce a new double-stranded polynucleotide molecule. This series of steps (denaturation, primer annealing, and primer extension) constitutes a PCR cycle. As cycles are repeated, the amount of newly synthesized polynucleotide increases geometrically because the newly synthesized polynucleotides from an earlier cycle can serve as templates or synthesis in subsequent cycles. Primer oligonucleotides are typically selected in pairs that can anneal to opposite strands of a given double-stranded polynucleotide sequence so that the region between the two annealing sites is amplified.
The temperature of the reaction mixture must be varied during each PCR cycle, and consequently varied many times during a test. For example, denaturation of DNA typically takes place at about 90xc2x0-100xc2x0 C., annealing a primer to the denatured DNA is typically performed at about 40xc2x0-60xc2x0 C., and the step of extending the annealed primers with a thermostable DNA-polymerase is typically performed at about 70xc2x0-75xc2x0 C. Each of these steps may have an optimal temperature.
Examples of PCR are disclosed in U.S. Pat. No. 4,683,202, U.S. Pat. No. 4,965,188 and U.S. Pat. No. 5,038,852.
Apparatus in which a temperature gradient is generated across a gradient block is described in U.S. Pat. No. 5,525,300. Multiple reaction mixtures may be held in wells on the gradient block. In preferred embodiments, the gradient block is integrated into a thermocycler used for nucleic amplification reactions.
U.S. Pat. No. 4,981,801 describes an apparatus for carrying out enzymatic cycling reactions including a turntable, a number of reaction vessels arranged in the turntable around the periphery and means to circulate antifreeze liquid through the reaction tank. Heaters and refrigerators are provided in order to obtain variations in temperature.
An apparatus to detect and enumerate a particulate analyte in a liquid sample comprising a filter element in a holder and means to heat and control the temperature of the filter element, is disclosed in WO 94/21780 of R.G.L. Wheatcroft and W. B. Berndt.
A method for detection and discrimination of multiple analytes using fluorescent technology is disclosed in U.S. Pat. No. 5,723,294.
EP 0 723 812 describes a thermal cycling reaction apparatus having a reactor with a reactor body made of a thin heat conductive plate and having a cavity as reaction chamber.
Additional apparatus and methods for conducting thermocycling reactions, especially polymerase chain reactions, in a rapid automated and controlled mariner would be beneficial.
Accordingly, one aspect of the present invention provides rotary thermocycling apparatus, especially for biochemical reactions, comprising:
(a) a plurality of stations for receiving samples in a flat-bottomed container, each station having a flat heated plate on which said container is placed and having means to independently control said heated plate at a pre-determined temperature;
(b) means to move each said flat-bottomed container from one station to another station in a pre-determined sequence;
(c) at least two of said stations having a heating unit adapted to be lowered over a container located on said station, each heating unit being comprised of a section with a flat lower surface that is adapted to be lowered into said container close to but not in contact with the sample in said container; and
(d) at least one station, in addition to the stations of (c), having a spray unit adapted to spray a liquid reagent(s) into a container located at said one station.
In a preferred embodiment of the invention, said station of (d) is adapted for removal of a cover plate from said container prior to activation of the spray, and for replacement of the cover plate after said spray has terminated.
In another embodiment, the container is a flat-bottomed container of a dimension less than that of t e station.
In a further embodiment, the heating is comprised of a section with a flat lower surface that is adapted to be lowered into said container close to but not in contact with the sample in said container, especially into contact with a cover plate therein.
In a still further embodiment, the apparatus has a programmer for controlling at least (i) the temperature at each station, (ii) the dwell time in each station, (iii) the duration and timing of the spray, (iv) the number of sequential cycles for the biochemical reaction.
In another embodiment, the container is adapted to receive a filter having the sample thereon, and to receive a cover plate over said filter.
In yet another embodiment, the apparatus is programmable and automated.
In a further embodiment it, the apparatus is adapted to process more than one sample at a time, up to one for each station in the apparatus.
In another embodiment, the heating units are on pistons that are lowered into the containers.
Another aspect of the present invention provides a method for a sequential reaction, especially a sequential biochemical reaction, at different temperatures, comprising:
(a) placing a sample in a flat-bottomed container;
(b) sequentially cycling said sample through predetermined changes in temperature by placing said flat-bottomed container on flat heated plate at each said temperature for a predetermined period of time;
(c) spraying said sample with at least one liquid reagent;
(d) controlling (i) the temperature at each station, (ii) the dwell time in each station, (iii) the duration and timing of the spray(s), and (iv) the number of sequential cycles for the biochemical reaction, said controlling of the temperature is at least two stations and includes the step of lowering a heating unit over the containers at said at least two stations, each heating unit being comprised of a section with a flat lower surface that is adapted to be lowered into said container close to but not in contact with the sample in said container.
In a preferred embodiment of the method of the invention, a biochemical sample is located on a filter, membrane, microtitre container or microscope slide in said container, especially a filter.
In another embodiment, the method is programmable and automated.
In a still further embodiment, the sample is subjected to a pretreatment prior to the method for the sequential reaction.
In a further embodiment, a spacer is placed on the filter and a cover plate is placed on the spacer.
In a further embodiment, the reaction is a polymerase chain reaction.
In another embodiment, the reaction is for detection of specific DNA sequences.
In a further embodiment, the sample is subsequently subjected to a photochemical detection process, especially fluorescence, to detect product of the reaction, and in particular to electronic recording thereof e.g. using a video camera.
A further aspect of the present invention provides rotary thermocycling apparatus especially for biochemical reactions, comprising:
(a) a plurality of stations for heating samples in a flat-bottomed container at predetermined temperatures;
(b) means to move each said flat-bottomed container from one station to another station in a pre-determined sequence; and
(c) at least one station having a spray unit adapted to spray liquid reagent(s) into a container located at said one station.
Another aspect of the present invention provides a method for a sequential biochemical reaction at different temperatures, comprising placing a biochemical sample on a filter and sequentially cycling said biochemical sample through predetermined changes in temperature by heating said filter on a sequence of flat heated plates for a predetermined period of time.
A further aspect of the invention provides a method for a sequential biochemical reaction at different temperatures, comprising placing a biochemical sample on a filter and sequentially cycling said biochemical sample through predetermined changes in temperature, at least one step in the sequence involving spraying the sample with liquid reagent(s).